ATP-binding cassette (ABC) transporters are ubiquitous transmembrane protein complexes that couple ATP binding and hydrolysis to the movement of substances across the membrane. More than a dozen human diseases have been traced to ABC proteins including cystic fibrosis, Startgardt disease and Tangier disease. Over-expression of some ABC transporters in tumor cells confers drug resistance by pumping the drugs out of the cells, a phenomenon that becomes one of the major barriers to effective treatment of cancer. We propose to study the molecular mechanism underlying the active transport process mediated by ABC proteins, using the E coli maltose transporter (MalFGK2) as a model system. Specifically, we plan to: (1) determine the crystal structures of the ATPase subunit, MalK, in different catalytic stages of the ATP hydrolysis; (2) to study biochemically and physically the mechanism underlying inducer exclusion, in which Enzyme IIAglc binds to MalK and inhibits maltose transport when glucose is present in the medium; (3) to reveal the molecular interface between MalK and the transmembrane subunits using various biochemical and physical methods; and (4) to determine the crystal structure of the entire transport complex in a transition-state-like intermediate form. At the completion of the proposed studies, we expect that we will have gained new knowledge on how the ATPase activity of the transporter is regulated, how ATPase subunits interact with the transmembrane subunits, and what happens during the maltose transport cycle. Because all members of ABC transporter family share a common domain organization and high degree of sequence similarity, the knowledge of the E. coli maltose transport mechanism will advance our understanding of how conformational changes of ABC transporters facilitate the transport processes.